11.2. BACKGROUND AND SIGNIFICANCE A number of independent pieces of evidence point to the selectivity filter as a region with a great deal of influence over the gating behavior of a channel, not only in regards to C-type inactivation but also in terms of on-off transitions during activation gating. The effect of certain permeant ions on gafing is have been well documented [1-4]. Ions with long occupancy fimes (Rb+, Cs+, NH4+) tend to stabilize the open state through a "foot in the door" effect on the gate, yet the only region of channel-ion interacfion occurs at the selecfivity filter. Additionally, channels seem to populate sub-conducting states on the way to the open state [5-7], and these sub-conducting states show altered selectivity. Unnatural amino acid mutagenesis targeted to the signature sequence of an inward rectifier K channel revealed dramatic consequences upon rapid gating transitions [8], again, pointing to the selectivity filter as a contributor to the gating process. Structurally, there is eariy evidence of subtle conformational changes in regions flanking the selectivity filter, and these changes appear only under conditions that favor channel opening [9]. KcsA undergoes C-type inacfivation similar to other biologically important K+ channels [10, 11]. After a transition to acidic pH, the lower gate at the inner-helical bundle opens and imparts conformational changes around the selectivity filter. This conformational wave leads to C-type inactivation. Nonetheless, a demonstration of the role of the selectivity filter in influencing activation gating requires additional structural approaches. Three recent developments in the Perozo lab have opened an interesting window of opportunity to further analyze the role of the different moving parts of a channel on gating. First, as stated above, we have identified a C-type inactivation mechanism in KcsA, found and characterized KcsA mutants around the selectivity filter.